Method for auxiliary identification of inbred line of wuzhishan miniature pig and its special primer

ABSTRACT

An auxiliary method for identification of WZSP inbred line and its special primers are provided. The method is to test if DNA at site 1273 from 5′ end of genomic DNA SEQ ID NO: 1 in test pig is A or G, measure the test pig genotype is GG, GA or AA; GG or AA genotype is homozygote that DNA at site 1273 is G or A; GA genotype is their heterozygote; test pig of GG genotype is candidate for the WZSP inbred line; test pigs of GA and AA genotypes are not WZSP inbred line. The method can be applied to breed WZSP inbred line, can preliminarily screen all the pigs in test pig group, eliminate non WZSP inbred line, find out candidate WZSP inbred line, combine with other methods for further confirmation. The method can also be used to judge whether WZSP on market is counterfeiting or not.

TECHNICAL FIELD

This invention relates to a method for auxiliary identification of WUZHISHAN miniature pig (WZSP) inbred line and its special primers.

TECHNICAL BACKGROUND

In early period of China's reform and opening up, the researcher, when studying in West Germany in 1983-1984, had a great privilege to visit the West Germany Gottengen Miniature Pig Breeding Center, learnt that the miniature pig were widely applied to Animal Models of Human Diseases in many western countries, and it was also a great subject for the study of porcine embryonic, Gottengen Miniature Pig had been marketed in many countries and regions, therefore had the idea of introducing it into China for development and application. Professor Smith, A Gottengen Miniature Pig Breeding expert, is interested in Chinese MEISHAN pig. And he suggested that the West German Ministry of Agriculture put it as a special support of bilateral cooperation, the researchers also get support from Ministry of Agriculture after returning to China. But the plan cannot be realized for all kinds of reasons. During that period, we found a large number of specific animal species in the domestic after a large-scale animal species resource investigation, such as WZSP. But they are on the verge of extinction (see the Investigation Report in which Feng Weiqi researcher of the China Academy of Agricultural Sciences, Wang Zheng of Institute of Animal Husbandry of Guangdong Province and seventeen researchers are participant, July 1987). From the data, the miniaturization characteristics, genetic stability of WZSP are much better than West Germany Gottengen Miniature Pig, and its prospects for application development will be quite good.

For 20 years of efforts since 1987, and under the support from the Ministry of Agriculture, Science and Technology, National Natural Science Foundation, and others, 15 million yuan has been invested in total. Chinese Academy of Agricultural Sciences animal husbandry institute use WZSP, which size is small, endangered species, isolated and breeded the first inbred pig in the world, and they has received the 20^(th) generation group (F₂₀). Wu Zhishan Miniature pig species subjects to inbred breeding more than 10 generations from full sib families, offspring-parent or three generations of WZSP and creates WZSP inbred group. And the experiment proves that it is highly homozygous in genes and genetically very stable. It is named WZSP inbred-line (ZL02149030.9). Cultivated inbred has a docile temperament, clear genetic background, highly homozygous gene, black and white in color, size and coat color consistency than the original breeding herd, and it has small size, the genetic stability and other advantages than domestic and foreign varieties. It is new animal genetic resources, and it has been listed as a national conservation farms (C1101001). A deep-level, multi-faceted research and exploitation and utilization of human Comparison medicine, such as Burns Skin Transplantation, Model Cardiovascular, Oral Tooth Diseases, xenotransplantation and so on, is made. The result indicates: it is not only good material for breeding pigs and high-grade meat processing, but also is the best choice for experimental animal models. WZSP inbred line will play a special role and plans an unmeasurable role in human medical research, xenotransplantation research.

INVENTION DISCLOSURE

The purpose of this invention is to provide an auxiliary identification method for WZSP inbred line and its special primers.

The present invention provides a auxiliary identification method for WZSP inbred line and/or NON WZSP line. It is to test deoxyribose nucleotide at site 1273 from 5′ end of genomic DNA SEQ ID NO: 1 (GenBank Accession Number DQ406743) in test pig is A or G, determine the test pig genotype is GG, GA or AA; GG genotype is homozygote that the deoxyribose nucleotide at site 1273 of SEQ ID NO: 1 is G; AA genotype is homozygote that the deoxyribose nucleotide at site 1273 of SEQ ID NO: 1 is A; GA genotype is their heterozygote; SEQ ID NO: 1

test pig of GG genotype is candidate for the WZSP inbred line;

test pigs of GA genotype and AA genotype are not WZSP inbred line.

The method used to determine the deoxyribose nucleotide at site 1273 from 5′ end of SEQ ID NO: 1 (GenBank Accession Number DQ406743) in test pig is G or A includes the following steps: genomic DNA extraction, PCR, enzyme cut and electrophoresis.

The primer pair for the PCR amplification meet the following conditions: product of PCR amplification using the pig genomic DNA as template contains the deoxyribose nucleotide at site 1273 from 5′ end of SEQ ID NO: 1 (GenBank Accession Number DQ406743) of the test pig.

Specifically, the primer pair for the PCR amplification is primer pair consisting of nucleotide set forth in SEQ ID NO: 2 and nucleotide set forth in SEQ ID NO: 3.

The restriction enzyme used in enzyme cut is TasI. The electrophoresis is specifically agarose gel electrophoresis. GG genotype pigs, PCR-RFLP testing (TasI enzyme cut) have four electrophoresis band: 357 bp, 204 bp, 134 bp and 67 bp in sequence length, respectively; AA genotype pigs, PCR-RFLP (TasI enzyme cut) have five electrophoresis band: 82 bp, 275 bp, 204 bp, 134 bp and 67 bp in sequence length, respectively; GA genotype pigs, PCR-RFLP (TasI enzyme cut) have six electrophoresis band: 357 bp, 82 bp, 275bp 204 bp, 134 bp and 67 bp in sequence length, respectively.

This invention is also protect a primer pair for supplementary identification of WZSP inbred line and/or non WZSP inbred line, the primer pair meet the following conditions: product of PCR using pig genomic DNA as template contains deoxyribose nucleotide at site 1273 from 5′ end of SEQ ID NO: 1.

The primer pair is specifically the primer pair consisting of nucleotide set forth in SEQ ID NO: 2 and nucleotide set forth in SEQ ID NO: 3.

The primer pair can be applied in the preparation of kit for assistant identification of WZSP inbred line and/or non WZSP inbred line.

This invention is also to protect a kit for auxiliary identification of WZSP inbred line and/or non WZSP inbred line, containing the primer pair mentioned above. The kit also includes PCR reagent and electrophoresis reagents.

The kit or the primer pair can be applied to in assistant identification of WZSP inbred line and/or non WZSP inbred line.

The kit, the primer and the method can be applied in pig breeding.

In the kit, the primer, the method and the uses, the WZSP inbred line can be F₁₃ to F₂₀ generation.

DESCRIPTION OF FIGURES

FIG. 1 is Restriction endonuclease map of three genotypes samples; M: DNA molecular weight standard (100-1500 bp ladder).

FIG. 1

THE BEST MODE FOR CARRYING OUT THE INVENTION

The following examples are used for better understanding of the invention, but not to limit the invention. The experimental methods of the following examples are conventional methods, unless noted otherwise. Without special instructions, the test materials used in the following examples, all get from conventional biochemical reagents shop.

WZSP inbred line (Li Kai; Mou Yulian; Han Jianlin; Yang Shulin; Liu Lan; Yuan Xinxu; Guo Yong; Feng Shutang, Study on Genetic Variation of Inbred Families of WZSP Using Microsatellite DNA Loci, Chinese Journal of Animal and Veterinary Sciences, 3^(rd) 2009).

Example 1, discovery of G1273A polymorphisms of Pnas-4 gene

The pig used in experiment: 42 Guizhou Miniature Pigs, 49 Guangxi Bama Miniature Pigs and 42 WZSP inbred line.

1. PCR Amplification

According to the second intron sequence of Pnas-4 genes (GenBank Accession Number DQ406743) (SEQ ID NO: 1 of sequence listing SEQ ID NO: 1) a primer pair is designed as follows:

Upstream primer (the SEQ ID NO: 2 of sequence listing): 5′-CTAGAACCACTCAAACCAAGCAGC-3′;

Downstream primer (The SEQ ID NO: 3 of sequence listing): 5′-ATCAGGCAGGTAAAAGGATAACGG-3′.

Using the above primer pair, with pig genomic DNA as being a template for PCR amplification.

Polymerase chain reaction (PCR) system: 2.0 μL 10× reaction buffer, 1.6 μL MgCl₂ (2.5 mmol/L), 1 μL upstream primer (10 μmol/L), 1 μL downstream primer (10 μmol/L), 0.4 μL. dNTPs (10 mmol/L), 0.2 μL Taq enzymes, 1 μL templates, ddH₂O constant volume to 20 μL.

PCR amplification program: 95° C. 3 min, 30 cycles (94° C. 20 s, 62.5° C. 30 s, 72° C. 30 s), finally 72° C. extensions for 3 min.

Amplification product evaluation is made by 1.5% AGAR gel electrophoresis.

2. RFLP Anlyse

Endonuclease Reaction system (10 μL): 1× buffer 1 μL, PCR products 5 μL, restriction enzymes TasI 0.5 μL (5U), and use H₂O to be complemental to 10 μL.

After enzyme cut for eight hours, 1.5% agarose gel electrophoresis is used to observe the results of enzyme cut.

Enzymes cut products presented three band types. Part of samples show four electrophoresis band, the sequence's length is respectively 357 bp, 204 bp, 134 bp and 67 bp; Part of samples show five electrophoresis band, the sequence's length is respectively 82 bp, 275 bp, 204 bp, 134 bp and 67 bp; The remaining samples show six electrophoresis band, the sequence's length is respectively 357 bp, 82 bp, 275 bp, 204 bp, 134 bp and 67 bp.

3. The Cloning Sequencing and Sequence Analysis

According to the PCR-RFLP analysis result, PCR amplification of sample displaying different electrophoresis bands is respectively recovered and purified with agarose gel recycling kit (Tiangen biochemical technology Co., LTD.). After the recovered DNA fragment is ligated to vector pGEM-T (Promega company), referring to Cohen method (Proc Natl Acad Sci, 69:2110), the ligation product is used to transform E.coli DH5α competent cells. According to carboxybenzylepenicillin resistance markers, positive clone is screened, the recombination plasmids containing recovered fragment are obtained. With T7 and SP6 promoter sequences on this recombination as primer, the nucleotide sequencing is made. The sequencing results show that the fragment lengths of different samples are all 762 bp, there exists only one deoxyribose nucleotide difference (G/A). This deoxyribose nucleotide is the deoxyribose nucleotide at site 1273 from 5′ end of GenBank Accession Number DQ406743 , named G1273A. Among them, the nucleotide sequence of the amplification fragment that deoxyribose nucleotide at site 1273 is G is set forth in SEQ ID NO: 1 OF sequence listing.

According to sequencing result and PCR-RFLP result, the genotype is defined as follows:

If the alleles at this site is G, its homozygote genotype is GG, PCR-RFLP testing (TasI enzyme cut) has four electrophoresis bands, the sequence's length is 357 bp, 204 bp 134 bp and 67 bp, respectively;

If the alleles at this site is A, its homozygote genotype is AA, PCR-RFLP testing (TasI enzyme cut) has five electrophoresis bands, the sequence's length is 82 bp, 275 bp, 204 bp 134 bp and 67 bp, respectively;

The heterozygote genotype for this site is GA, PCR-RFLP (TasI enzyme cut) testing has six electrophoresis bands, the sequence's length is 357 bp, 82 bp, 275 bp 204 bp, 134 bp and 67bp, respectively.

Using the primer of the invention to PCR amplify genomic DNA of test pig, 762 bp fragment is obtained. If the deoxyribose nucleotide at site 1273 from 5′end of GenBank Accession Number DQ406743 is A, after enzymes cut with restriction enzymes TasI, 357 bp fragment is divided into 82 bp and 275 bp fragments.

See FIG. 1 for restriction endonuclease map of three genotypes samples.

See table 1 for the detection results of different varieties of pig genotypes.

TABLE 1 TasI polymorphism test results of Pnas-4 gene of different varieties of pig Genotype Allele Genotype Frequency Frequencies Variety Number GG GA AA GG GA AA G A Guizhou Mini Pig 42 31 11 0 0.738 0.262 0.000 0.87 0.13 Guangxi Bama Mini 49 2 14 33 0.041 0.286 0.673 0.18 0.82 Pig WZSP 42 42 0 0 1.000 0.000 0.000 1.000 0.00

Results show that the WZSP inbred line exist only GG genotype and G alleles and other varieties of miniature pig exist A allele.

This invention found a polymorphism e site, WZSP has only G allele in this polymorphism site and other varieties of pigs, exist A alleles. So this polymorphism site can be used to screen test pigs. If the test pig is GG genotype, it can be used as candidate WZSP line, if the test pig is GA genotype or AA genotype, it is certainly not WZSP inbred line. The method of the present invention can be applied to breed WZSP inbred line. All the pigs in test pig population can subject to preliminary screening in advance, the non WZSP is eliminated, the candidate WZSP inbred line is found out, other methods are combined for further confirmation. The present invention can also be used judge whether WZSP on market is counterfeiting or not.

Example 2, Use of G1273A polymorphisms of gene Pnas-4

Pig ear skin tissue samples are all collected from WZSP inbred line from the Chinese academy of agricultural sciences of the animal husbandry and veterinary institute of Beijing WZSP, which contain F₁₃ (generation) 15 (pigs), F₁₄ 13, F₁₅ 11, F₁₆ 14, F₁₇ 14, F₁₈ 14, F₁₉ 18, F₂₀ 23, all the samples treated with 75% ethanol, and freezed in −20□, prepared to extract DNA.

1. PCR Amplification

Primer is as follows:

Upstream primer (SEQ IN NO: 2 of the sequence listing): 5′-CTAGAACCACTCAAACCAAGCAGC-3′; Downstream primer (SEQ IN NO: 3 of the sequence listing): 5′-ATCAGGCAGGTAAAAGGATAACGG-3′.

Using the above primer pair, with pig genomic DNA as being a template for PCR amplification.

Polymerase chain reaction (PCR) system: 2.0 μL 10× reaction buffer, 1.6 μL MgCl₂ (2.5 mmol/L), 1 μupstream primer (10 μmol/L), 1 μL downstream primer (10 μmol/L), 0.4 μL dNTPs (10 mmol/L), 0.2 μL. Taq enzymes, 1 μL templates, ddH₂O constant volume to 20 μL.

PCR amplification program: 95° C. 3min, 30 cycles (94° C. 20 s, 62.5° C. 30 s, 72° C. 30 s), finally 72° C. extensions for 3min.

Amplification product evaluation is made by 1.5% AGAR gel electrophoresis.

2. RFLP Analysis

Endonuclease Reaction system (10 μL) as follows: 1× buffer 1 μL, PCR products 5 μL, restriction enzymes TasI 0.5 μL (5U), use H₂O to be complemental to 10 μL.

After enzyme cutting for 8 hours, the enzyme cutting result is detected with 1.5% agarose gel electrophoresis, and through the gel imaging system the best quality images are obtained, genotypes are recorded.

See results in table 2.

TABLE 2 genotype test results of different generations of WZSP inbred line Genotype Allele Gener- Num- Genotype Frequency Frequencies ation ber GG GA AA GG GA AA G A F₁₃ 15 15 0 0 1.000 0.000 0.000 1.000 0.000 F₁₄ 13 13 0 0 1.000 0.000 0.000 1.000 0.000 F₁₅ 11 11 0 0 1.000 0.000 0.000 1.000 0.000 F₁₆ 14 14 0 0 1.000 0.000 0.000 1.000 0.000 F₁₇ 14 14 0 0 1.000 0.000 0.000 1.000 0.000 F₁₈ 14 14 0 0 1.000 0.000 0.000 1.000 0.000 F₁₉ 18 18 0 0 1.000 0.000 0.000 1.000 0.000 F₂₀ 23 23 0 0 1.000 0.000 0.000 1.000 0.000

The results show that all are only GG genotype in F₁₃ to F₂₀ generations.

INDUSTRIAL APPLICATION

This invention discovers that WZSP inbred line is homozygous at G1273A polymorphic site, only exits GG genotype. But other varieties of miniature pigs exist GA, AA genotype. So this polymorphism site can be used to screen test pigs. If the test pig is GG genotype, it can be used as candidate WZSP line, if the test pig is GA genotype or AA genotype, it is certainly not WZSP inbred line. The method of the present invention can be applied to breed WZSP inbred line. All the pigs in test pig population can subject to preliminary screening in advance, the non WZSP is eliminated, the candidate WZSP inbred line is found out, other methods are combined for further confirmation. The present invention can also be used judge whether WZSP on market is counterfeiting or not. 

1. An auxiliary method for identification of Wu Zhishan miniature pig (WZSP) inbred line and/or non WZSP inbred line, comprising: testing if a deoxyribose nucleotide corresponding to a deoxyribose nucleotide at site 1273 from 5′ end of genomic DNA SEQ ID NO: 1 in test pig is A or G; and measuring the test pig genotype is GG, GA or AA; wherein said GG genotype is homozygote that the deoxyribose nucleotide corresponding to site 1273 from 5′ end of genomic DNA SEQ ID NO: 1 is G; said AA genotype is homozygote that the deoxyribose nucleotide corresponding to site 1273 from 5′ end of genomic DNA SEQ ID NO: 1 is A; said GA genotype is their heterozygote; test pig of GG genotype is candidate for the WZSP inbred line; test pigs of GA genotype and AA genotype are not WZSP inbred line.
 2. The method according to claim 1, wherein said testing if a deoxyribose nucleotide corresponding to deoxyribose nucleotide at site 1273 from 5′ end of genomic DNA SEQ ID NO: 1 in test pig is G or A comprises one or more selected from the group consisting of: extracting genomic DNA of the test pig; performing a polymerase chain reaction (PCR) with the extracted genomic DNA and a pair of primer; cutting DNA with a restriction enzyme; and electrophoresing DNA.
 3. The method according to claim 2, wherein the primer pair for the PCR meet the following conditions: a product of PCR using the test pig genomic DNA as template contains the deoxyribose nucleotide corresponding to deoxyribose nucleotide at site 1273 from 5′ end of genomic DNA SEQ ID NO: 1 of the test pig.
 4. The method according to claim 3, wherein the primer pair for the PCR is a primer pair consisting of nucleotide set forth in SEQ ID NO: 2 and nucleotide set forth in SEQ ID NO:
 3. 5. The method according to claim 2, wherein the restriction enzyme used in enzyme cut is TasI; the electrophoresis is agarose gel electrophoresis.
 6. The method according to claim 1, wherein the WZSP inbred line is F₁₃ to F₂₀ generation.
 7. Primer A primer pair for auxiliary identification of WZSP inbred line and/or non WZSP inbred line, wherein the primer pair are configured to correspond to portions of a pig genomic DNA in a polymerase chain reaction (PCR) so as to amplify a product of PCR which comprises deoxyribose nucleotide corresponding to deoxyribose nucleotide at site 1273 from 5′ end of genomic DNA SEQ ID NO:
 1. 8. The primer pair according to claim 7, wherein the WZSP inbred line is F₁₃ generation to F₂₀ generation.
 9. A primer pair consisting of nucleotide set forth in SEQ ID NO: 2 and nucleotide set forth in SEQ ID NO:
 3. 10. A kit for auxiliary identification of WZSP inbred line and/or non WZSP inbred line, comprising the primer pair according to claim
 7. 11. The kit according to claim 10, wherein the WZSP inbred line is F13 generation to F20 generation.
 12. (canceled)
 13. (canceled)
 14. (canceled)
 15. (canceled)
 16. (canceled)
 17. A kit for auxiliary identification of WZSP inbred line and/or non WZSP inbred line, comprising the primer pair according to claim
 9. 